Immune checkpoint inhibitor therapy (ICT) unleashes the immune system resulting in durable remissions in up to 50% of patients with previously incurable cancers, such as metastatic melanoma. But the host factors that modulate or dictate ICT response are poorly understood. Recent, preclinical data suggests that specific gut microbiota are required for optimizing response to ICT. These commensal gut microbiota augment host adaptive immune responses, specifically host immune cells, needed for anti-cancer activity. We recently showed that adult melanoma patients who respond to ICT have a distinct gut microbiota signature compared to patients with progressive disease. In preliminary data, we demonstrated that precision probiotic therapy (using the specific bacteria that we identified in our clinical/translational study) augmented the efficacy of ICT in mice with melanoma whereas a probiotic commonly found in yogurt did not. Microbiota dictated a reduction in PD-1 expressing CD4 and CD8 T cells in mesenteric lymph nodes (MLNs) following ICT. Of note, these specific gut microbiota had a greater number of protein sequences homologous to human melanoma neoantigens when compared to the ineffective yogurt probiotic. Further, these gut microbiota induced CD4 and CD8 T cell production of IFN-gamma and Granzyme B. Interestingly, mice without draining lymph nodes in the gut did not respond to ICT. ICT promoted gut microbiota translocation into MLNs. Finally, oral administration of heat-killed gut microbiota did not augment ICT efficacy, suggesting that live bacteria are required for augmenting ICT. Therefore, our central hypothesis is that only gut microbiota that have both high immunogenic potential and the ability to translocate into the abdominal draining lymph nodes will facilitate activation/education of immune cells and thus augment ICT efficacy. To test this hypothesis, we will pursue the following three specific aims. First, we will further define both gut microbiome and tumor genetic differences in melanoma patients who respond to ICT compared to those with disease progression. Second, we will identify environmental and microbial factors that dictate gut microbiota translocation into mesenteric lymph nodes. Third, we will elucidate the mechanisms by which gut microbiota modulate host immune cell anti-tumor response by performing in vitro functional immune cell assays to determine if and how specific gut microbiota prime immune cells and in vivo studies to identify the specific immune cells recruited by gut microbiota. These studies will lay the groundwork for the following innovations: 1) a novel mechanism by which gut microbiota activate immune defenses against cancer and 2) a novel approach (precision probiotics) for optimizing ICT efficacy in advanced cancer patients.